Non-linear scanning switch-off microscopy for super-resolution fluorescence imaging

Abstract Super-resolution (SR) microscopy provides a revolutionary approach to study cells and animals by breaking the diffraction limit of optical imaging. However, the popularity of the super-resolution microscope in biological sciences remains to be impeded by the high cost of hardware and/or the complexity of software. Here, we present a conceptually different non-linear scanning switch-off microscopy (nSSM) that exploits the omnipresent switch-off effect of fluorophores to enable super-resolution imaging beyond the diffraction limit. We develop a theoretical model of nSSM and experimentally implement the nSSM scheme with an unmodified confocal microscope. We also release a free code for the automatic reconstruction of super-resolution images. By measuring the PSF of the imaged DNA origami nanostructure and mammalian cytoskeleton structures, we demonstrate an SR resolution of ~ 100 nm that excels the optical resolution limit by over two folds. We further show the generality of nSSM using a range of commercially available fluorescent dyes and proteins to realize SR imaging in various settings. This nSSM methodology may in principle empower any confocal microscope to implement SR imaging to promote biological research.